Method and apparatus for image scanning

ABSTRACT

A method of estimating an in-focus level of a target in an image scanning apparatus is provided, wherein the image scanning apparatus comprises a first line scan detector configured to obtain one or more image scan lines of the target and a second line scan detector configured to obtain one or more focus scan lines of the target. The method comprises obtaining at least one image scan line of the target using the first line scan detector, each at least one image scan line being obtained at a respective focus level; obtaining at least one focus scan line of the target using the second line scan detector, each at least one focus scan line being obtained at a respective focus level; calculating at least one focus parameter using at least one focus scan line; and estimating a nominal in-focus level of the target using the calculated focus parameter(s).

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for imagescanning, and in particular, but not limited to, the use of a virtualmicroscope.

BACKGROUND TO THE INVENTION

FIG. 1 illustrates a typical arrangement of a virtual microscope usedfor image scanning as is known in the prior art. The arrangementcomprises an imaging lens 1 which focuses light originating from a slide6 onto a line scan detector 2. The imaging lens and the line detectortogether make up an imaging system. As the detector 2 is a line scandetector, the image area 7 is a line. In order to produce an extendedimage over a larger area of the slide 6, the slide is moved relative tothe imaging lens and line scan detector, as indicated by arrow 8. Inthis sense the slide is “scanned” by the line scan detector.

The line scan detector is typically used to image a sample prepared uponthe slide. The sample may be a biological specimen for example.Typically, the sample to be imaged will have an inhomogeneous surfacetopography with a focus variation greater than the depth of field of theimaging system. Typically, a single scan of the slide will beapproximately 1 mm wide and between 2 mm and 60 mm long. Over the scaleof 1 mm, the focus of the sample very rarely exceeds the depth of focusof the imaging system (typically approximately 1 μm). However, overlarger distances such as 20 mm, the change of focus of the sample canexceed the depth of field of the imaging system. There is therefore theproblem that the output image produced by a line scan detector whilescanning a sample is likely to have areas which are in focus and areaswhich are out of focus, due to changes in the surface topology of thesample. This is unacceptable, especially in cases where accurateanalysis of the sample is required.

There have been various attempts made to overcome this problem. Forexample, U.S. Pat. No. 7,518,652 discloses the use of a focus mapwherein the adjustment of focus of the imaging system during the scan ispredetermined. However, this requires the whole sample to be analysedbefore the scan can commence which is very time consuming, or onlyparticular points on the sample are taken and therefore areas betweenthe points are unlikely to have good focus.

U.S. Pat. No. 7,485,834 discloses temporarily changing the focus of theimaging lens during scanning of the sample to see if there is a betterfocus position. However, as the scan speed of the sample increases, thismeans that there is less time to move the imaging lens in search of abetter focus position. This means that either the scanning speed has tobe kept below a certain speed, or the change of position of the imaginglens has to occur over more imaging lines, which is more difficult tointerpolate the image across. Both of these scenarios are 1-0undesirable.

U.S. Pat. No. 7,330,574 discloses a 2D imaging detector which is tiltedin the scanning direction such that the best-focus plane of the imagingsystem intercepts the surface of the sample during the scan. The sampleis moved one, or a small number of frames for each frame, therebybuilding up a 3D scan of the sample which can be used for focuscalculation. This is done before scanning, as if the process were to beperformed during the scan, the data rate required by the 2D scannerwould be much greater.

There is therefore a requirement to improve the focussing of a sampleduring an imaging scan, such that the sample can be scanned quickly, andin focus.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there isprovided a method of estimating an in-focus level of a target in animage scanning apparatus, wherein the image scanning apparatus comprisesa first line scan detector configured to obtain one or more image scanlines of the target and a second line scan detector configured to obtainone or more focus scan lines of the target, the method comprising:obtaining at least one image scan line of the target using the firstline scan detector, each at least one image scan line being obtained ata respective focus level; obtaining at least one focus scan line of thetarget using the second line scan detector, each at least one focus scanline being obtained at a respective focus level; calculating at leastone focus parameter using at least the at least one focus scan line; andestimating a nominal in-focus level of the target using the calculatedfocus parameter(s).

Here, the term “level” can be seen to be analogous to “position”, suchthat the nominal in-focus level is the position of the focal plane ofthe image scanning apparatus when imaging the target. Preferably, thefirst line scan detector is operable to obtain an output image of thetarget which is desired to be in focus, and therefore the methodpreferably further comprises adjusting the focus level of the first linescan detector to the nominal in-focus level of the target. In otherwords, the position of the first line scan detector is adjusted suchthat the first line scan detector is in the focal plane of the imagescanning apparatus and the target is thus in focus.

The present invention advantageously enables the in-focus level of thetarget to be obtained quickly and easily, thus allowing in-focus imagesof the target to be obtained. This is particularly advantageous when thetarget is moved relative to the image scanning apparatus such that thefirst and second line scan detectors “scan” the target a line at a time.The target (which may be a biological specimen on a slide for example)may have a relief structure with varying topography which will alter itsin-focus level as it is scanned by the image scanning apparatus. Beingable to estimate the in-focus level quickly and accurately is thereforeadvantageous.

The at least one focus parameter is a means of relating a focus levelsof a line scan detector to a measure of how “in focus” an image scanline is at that particular focus level. The focus parameter may take anumber of forms, although preferably the focus parameter is a “focusmerit” value having a maximum value representing an in-focus level. Thefocus merit value maps how “in focus” the image scan lines are on to anumerical scale. For example, an image scan line which is perfectly infocus will have a focus merit value of “1”, whereas an image scan linewhich is perfectly out of focus will have a focus merit value of “0”.

Using a second line scan detector to obtain at least one focus scan linemeans that the in-focus level does not have to be found by moving thefirst line scan detector until the in-focus level is obtained. This isan extremely time consuming operation and is undesirable. For example,with the use of only one line scan detector, line images are“sacrificed” as the detector is moved until desired focus level isobtained. The use of the separate focus line scan allows a quickestimation of the in-focus level without the need for said “sacrificing”of line images.

The method of the present invention comprises calculating at least onefocus parameter using at least the at least one focus scan line. It isimportant that at least one focus parameter is obtained from the atleast one focus scan line in order to estimate the in-focus level. Insome embodiments the in-focus level may be estimated using only focusparameter(s) obtained from the focus scan line(s) without using theimage scan lines. This works best when the target is substantiallyhomogenous across its area imaged by the line scan detector. However,the in-focus level may also be estimated by comparison of a focusparameter calculated from the at least one focus scan line with a focusparameter calculated from the at least one image scan line. In someembodiments more than one focus parameter may be obtained using aplurality focus scan lines, whereas only one focus parameter may becalculated using a single image scan line. The opposite scenario mayalso be true. In other embodiments a plurality of focus parameters maybe calculated using a plurality of focus scan lines, and a plurality offocus parameters may be calculated using a plurality of image scanlines. A method may utilise any combination of the above possibilities.

The method may further comprise the step of calculating at least onefurther focus parameter using either said at least one image scan lineor a further said focus scan line. For example, a further focusparameter may be obtained from an image scan line. By ensuring that thefocus level of the at least one focus scan line is different to thefocus level of the at least one image scan line, this further focusparameter can be compared with the focus parameter calculated using afocus scan line in order to estimate the in-focus level. Preferably thefocus parameters are normalised (typically to the focus parametercalculated from the image scan line). This simultaneous calculation oftwo focus parameters at differing focus levels allows for a particularlyfast and accurate in-focus level estimation. Alternatively, a furtherfocus parameter may be obtained from a further focus scan line at adifferent focus level to that of the first focus scan line. The twofocus parameters from the focus scan lines can then be used to estimatethe in-focus level.

The calculating step may further comprise calculating at least one focusparameter for each of the first line scan detector and second line scandetector using the respective at least one image scan line and at leastone focus scan line. For example, a focus parameter may be obtained forthe first line scan detector using an image scan line and a focusparameter may be obtained for the second line scan detector using afocus scan line. These focus parameters may be compared in order toestimate the in-focus level. Alternatively for example, a plurality offocus parameters may be obtained for the second line scan detector and aplurality of focus parameters may be obtained for the first line scandetector. As a further example a plurality of focus parameters may becalculated for the second scan line detector and a single focusparameter calculated for the first line scan detector.

By ensuring that the focus level of the first line scan detector and thefocus level of the second line scan detector are different (for example,the second line scan detector may be placed at a shorter optical pathdistance from the target than the first line scan detector), the focusparameters calculated for the first and second line scan detectors canbe used to estimate the nominal in-focus level. The focus parameters maysimply be compared in order to estimate the in-focus level. For example,if the focus merit value of the focus scan line is less than the focusmerit value of the image scan line, the in-focus level is either abovethe first line scan detector (i.e. the first line scan detector ispositioned between the target and the in-focus level), or is already atthe in-focus level. Therefore, by using a second line scan detector at adifferent focus level to the first line scan detector, two comparablefocus parameters are obtained simultaneously and the in-focus level ofthe target can be estimated quickly.

The step of obtaining at least one focus scan line typically comprisesmodulating a focus level of the second line scan detector such that aplurality of focus scan lines are obtained at different focus levels. Insuch a case, a plurality of focus parameters are typicallycalculated—one for each of the plurality of focus levels of the secondline scan detector. These plurality of focus parameters are preferablynormalised to a focus parameter obtained from an image scan line andused to generate a “focus merit curve”, which plots the focus meritobtained at each focus level against the focus levels. The maximum ofthis curve can then be used to estimate the in-focus level of the targetand the first line scan detector is moved towards this maximum.Modulating the focus level of the second line scan detectoradvantageously removes the requirement for several line scan detectorsat different focus levels, which would decrease the amount of lightimpinging on the first line scan detector, thereby decreasing imagequality.

There are a number of ways of modulating the focus level of the secondline scan detector, which shall be discussed in more detail below.

The image and focus scan lines may be obtained from a common positionwithin a plane passing through the target and having a plane normaldefining an optic axis along which each of the first and second linescan detectors receive image information so as to produce the saidrespective image and focus scan lines. Image information may bereflected to one of the said line scan detectors using a beam splitter.This ensures that each of the first and second line scan detectorssimultaneously image the same spatial location on the target as the beamsplitter produces two images of the same spatial location on the target.A beam splitter may also be used to direct image information to a thirdline scan detector at a further focus level different to those of thefirst and second line scan detectors, such that a further focusparameter can be calculated. Using as beam splitter advantageously meansthat the calculated focus parameters are not affected by spatialvariations in the target, thereby simplifying the in-focus levelestimation and improving its accuracy. Other means of reflecting theimage information are envisaged, however.

Alternatively, the image and focus scan lines may be obtained fromdifferent positions in the target and wherein image information isobtained by the said first and second line scan detectors alongdifferent optic axes from the target. Although this means that the firstand second line scan detectors simultaneously image different spatialregions of the target, this does ensure that each detector is fullyilluminated (unlike with the use of a beam splitter), thereby improvingimage quality. This is particularly important for the output image fromthe first line scan detector.

The first and second line scan detectors may be located adjacent eachother, or alternatively image information may be reflected to one of thesaid line scan detectors using a mirror, such as a turning mirror. Asthe first and second line scan detectors receive image information alongdifferent optic axes from the target, use of the mirror to reflect lightto one of the line scan detectors advantageously does not reduce theillumination of the other line scan detector.

The method may further comprise rotating the mirror about a pointcentred upon the optic axis of the line scan detector to which it isreflecting image information (preferably the second line scan detector),so as to provide focus scan lines of the target at different focuslevels. In a similar manner as described above, here the focusparameters may be used to generate a focus merit curve plotting thefocus merit values of the focus scan lines at each focus level. The peakof the focus merit curve then provides the nominal in-focus level of thetarget and the first line scan detector is moved towards that maximum.

Alternatively, the mirror may be rotated about a point displaced fromthe optic axis of the line scan detector to which it is reflecting imageinformation. Advantageously, this provides a greater change in focuslevel with the same turning angle of the mirror.

If the target is moved relative to the image scanning apparatus (i.e.during a scan), the target is preferably moved in accordance with therotation of the mirror such that the focus line scans are obtained froma common location upon the target. This ensures that the focusparameters, such as focus merit values, are not affected by spatialvariations in the target. This allows for a more accurate estimate ofthe in-focus level.

As an alternative to rotating the mirror, the method may comprise movingthe second line scan detector with respect to the target so as to obtaina plurality of focus line scans at different focus levels. The movementof the second line scan detector is preferably to and fro along itsoptic axis. In a similar manner as described above, here the focusparameters may be used to create a focus merit curve by plotting thefocus merit values of the focus scan lines at each focus level. The peakof the focus merit curve then provides the estimated nominal in-focuslevel of the target.

As a further alternative, the method may comprise the step of modulatingthe focus level as a function of position across the scan line of thesecond line scan detector. For example, the second line scan detectormay be rotated about an axis perpendicular to its optic axis. Thisprovides a differential focus along the scan line of the detector, whichcan be used to calculate the focus parameter(s) for the second line scandetector. As an alternative, the second line scan detector may bepositioned at an angle to the optic axis such that each position on thesecond line scan detector is at a different focus level. A focusparameter may then be calculated at each focus level.

Modulating the focus level as a function of position across the scanline of the second line scan detector in order to estimate a nominalin-focus level of the target works well when the target is substantiallyhomogenous in space across the area (line) to be imaged by the linedetectors (i.e. there are no areas on the target which are particularlydetailed in comparison to the rest of the target, and the topography issubstantially constant). However, if the sample has an area of detailthat will only be imaged at one end of the first and second detectors,this can undesirably shift the estimated nominal in-focus level awayfrom the correct level. In order to counter this, the method may furthercomprise using image data from one or each of the at least one focus orimage scan lines to generate a detail parameter, and using the detailparameter in calculating the focus parameter(s). The focus parameter istherefore “normalised” to the level of detail in the target. The detailparameter is typically a nominal level of inhomogeneity within thetarget as a function of position on the line scan detector. For example,if there is a large amount of detail on the left side of the sample, thedetail parameter will peak at the left side of the detector. Preferablythe first line scan detector generates the detail parameter. Typicallythe detail and focus parameters are the same parameter, where the focusparameter is calculated from the focus scan line and the detailparameter is calculated from the image scan line.

The line scan detectors may be multi-channel detectors, with the methodfurther comprising calculating an in-focus level for different channelsof the detector. Typically the multi-channel detector will be an RGBdetector. Due to the different frequencies of coloured light, each ofthe RGB channels has a different focus level. This feature can be usedto evaluate a focus parameter for each channel and using one or more ofthe focus parameters for the channels in estimating the nominal in-focuslevel of the target. Evaluating a focus parameter for each channelprovides an increased number of data points, thereby improving theaccuracy of the estimated in-focus level.

If the target is moved relative to the image scanning apparatus, atemporal shift may be applied between the data from the scan lines ofthe first and second line scan detectors, wherein the temporal shift isa function of the relative movement between the target and the imagescanning apparatus. This advantageously ensures that the data from thescan lines of the first and second line scan detectors is from the samespatial location on the target. This improves the accuracy of thein-focus level estimation as the focus parameters (for example focusmerit values) are not affected by spatial variation on the target.

The image scan lines may be obtained from a number of locations upon thetarget so as to form a swathe. This is preferably performed by movingthe target relative to the first and second line scan detectors suchthat the detectors image the target one line at a time. The line scandetectors preferably each comprise a linear array of sensors in order toscan one line of the target at a time. The target is typically moved ina plane perpendicular to the optic axis of at least one of the line scandetectors. Preferably, the focus level of the first line scan detectoris adjusted to the nominal in-focus level in real time during theformation of a swathe such that the image scan lines within the swatheare obtained at different focus levels. This advantageously allows fast,in-focus scanning of a target. The scanning speed may be temporarilyslowed if necessary in order to allow time for the first line scandetector to adjust to the in-focus position. However, it is generallyassumed that the in-focus level will not substantially change over asmall number of image lines, so this is not always necessary.

According to a second aspect of the present invention there is providedimage scanning apparatus comprising: a first line scan detectorconfigured to obtain one or more image scan lines of a target; a secondline scan detector configured to obtain one or more focus scan lines ofthe target; and a processor configured to: obtain at least one imagescan line of the target at a respective focus level using the first linescan detector; obtain at least one focus scan line of the target at arespective focus level using the second line scan detector; calculate atleast one focus parameter using at least the at least one focus scanline; and estimate a nominal in-focus level of the target using thecalculated focus parameter(s).

Preferably, the image scanning apparatus further comprises a firstfocussing device configured to modify the focus level between the targetand the first line scan detector, and wherein the processor is furtherconfigured to operate the first focussing device to move the focus levelof the first line scan detector to the estimated nominal in-focus level.This ensures that once the in-focus level has been estimated, the firstline scan detector can be moved to said level in order that images ofthe target are in focus.

Preferably the image scanning apparatus further comprises a target stagefor retaining the target, imaging optics for causing an image of thetarget to be provided to the first and second line scan detectors, and adrive system for causing the first line scan detector to obtain imageinformation from different locations on the target. The imaging opticsmay for example comprise a lens for converging light rays originatingfrom the target on to the first and second line scan detectors. In acase where each of the first and second line scan detectors is arrangedto image a common location upon the target, the imaging optics mayinclude a beam splitter to direct part of the image information from thetarget to the first line scan detector and part to the second line scandetector. In a case where the first and second line scan detectors lieupon different respective optic axes of the imaging optics, the imagingoptics preferably includes a mirror arranged to direct part of the imageinformation from the target to one of the first or second line scandetectors.

The drive system is preferably operable to move the target with respectto the first and second line scan detectors such that the first andsecond line scan detectors receive image information from the wholetarget. For example, the drive system may be operable to move the targetstage, with the first and second line scan detectors and imaging opticsheld stationary; or may be operable to move the first and second linescan detectors and imaging optics, with the target held stationary.

Where the imaging optics comprises a mirror, the image scanningapparatus may further comprise a mirror drive adapted to rotate themirror so as to direct different image information to the said line scandetector. The rotation of the mirror means that focus scan lines atdifferent focus levels are obtained, and these are used in thegeneration of the focus parameter(s) of the respective line scandetector. Preferably, the mirror drive is operated in accordance withthe drive system such that the focus line scans are obtained from acommon location upon the target.

The image scanning apparatus may further comprise a detector driveadapted to move the second line scan detector to and fro along itsrespective optic axis. In a similar manner to the mirror drive, thismeans that focus scan lines at different focus levels are generated,which are used in the generation of the focus parameters.

Alternatively, the image scanning apparatus may further comprise adetector drive adapted to rotate the second line scan detector so as tomodulate the focus level as a function of position across the scan lineof the second line scan detector. Again, this generates a plurality offocus scan lines at different focus levels.

The apparatus may further comprise a third line scan detector forproviding further focus scan lines. Preferably the third line scandetector is positioned at a different focus level to the first andsecond line scan detectors, with each detector at a different focuslevel. Typically at least one focus parameter will be calculated for thethird line scan detector using the further focus scan lines. Thisprovides more data points to accurately estimate the in-focus level.

One or each of the first and second line scan detectors may be amulti-channel detector. Preferably the multi-channel detectors are RGBdetectors operable to detect red, green and blue light.

Typically the focus levels of the first and second line scan detectorsare independently controllable. The first and second scan line detectorsare also preferably identical such that the focus parameters are notaffected by differences in the detectors. Preferably the apparatus is avirtual microscope.

The skilled person will appreciate that the line scan detectorsdescribed in the first and second aspects may be replaced with anysuitable imaging detector.

According to a third aspect of the present invention there is provided acomputer program product comprising program code means adapted toperform the method according to the first aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described with reference to thefollowing drawings, in which:

FIG. 1 illustrates an image scanning apparatus as is known in the art;

FIG. 2 illustrates an image scanning apparatus according to a firstembodiment of the invention;

FIGS. 3A to 3D show focus merit curves according to the first embodimentof the invention;

FIGS. 4A to 4D show focus merit curves according to a second embodimentof the invention;

FIG. 5 illustrates an image scanning apparatus according a thirdembodiment of the invention;

FIGS. 6A to 6C show focus merit curves according to the third embodimentof the invention;

FIGS. 7A and 7B illustrate a detector layout according to a fifthembodiment of the invention;

FIG. 8 illustrates an image scanning apparatus according to a sixthembodiment of the invention;

FIG. 9 illustrates an image scanning apparatus according to a seventhembodiment of the invention;

FIG. 10 illustrates an image scanning apparatus according to an eighthembodiment of the invention;

FIG. 11 shows a focus merit curve according to the eighth embodiment ofthe invention;

FIG. 12 illustrates an image scanning apparatus according to a ninthembodiment of the invention;

FIG. 13 illustrates an image scanning apparatus according to a tenthembodiment of the invention;

FIG. 14 illustrates an image scanning apparatus according to an eleventhembodiment of the invention;

FIG. 15 illustrates a scan line according to the eleventh embodiment ofthe invention;

FIGS. 16A to 16C show focus merit and focus position curves according tothe eleventh embodiment of the invention; and

FIG. 17 shows focus merit and detail merit curves according to a twelfthembodiment of the invention.

DETAILED DESCRIPTION OF THE DRAWINGS

Throughout the following description, like reference numerals indicatelike parts. Features from an embodiment may be combined with featuresfrom any of the other embodiments.

FIG. 2 illustrates a schematic view of a first embodiment of a virtualmicroscope according to the present invention. The image scanningapparatus 100 of the first embodiment comprises an imaging line scandetector 2 and a focussing line scan detector 3. An image of the sample(not shown) is imaged through lens 1 onto the imaging line scan detector2. Typically the sample is moved relative to the apparatus 100 in aplane perpendicular to the optic axis of the imaging line scan detector2 such that the sample is imaged as a series of line scans. It isdesirable for the imaging line scan detector 2 to be placed in the focalplane of the imaging lens 1 such that the image is in focus throughoutthe scan. A beam splitter 6 is provided between the imaging lens 1 andthe imaging line scan detector 3 and divides the imaging beam into twoand produces a second image of the same spatial location of sample atco-conjugate plane 7. The focussing line scan detector 3 is positionedat a different focus level 8 to that of the imaging line scan detectorsuch that the imaging line scan detector 2 and the focussing line scandetector 3 produce image scan lines at different focus levels. In FIG. 2the focus level 8 is such that light travels further to the imaging linescan detector than the focussing line scan detector (the focus level 8is below co-conjugate plane 7), although the skilled person willappreciate that the focussing line scan detector 8 could also bepositioned above the co-conjugate plane 7.

A “focus merit” value is then calculated for both the imaging line scandetector 2 and the focussing line scan detector 3. The calculation istypically based on the sum of the square of the differences betweenadjacent pixels, although alternative calculation routines may be used,for example based on the power through a high-pass or band-passfrequency filter. The focus merit value is a measure of how in focus theimage scan lines obtained from the line scan detectors are and has amaximum value at an in-focus level. Such a value provides a numericalvalue which is dependent upon the amount of fine detail within the imageinformation, with a larger focus merit value indicating more fine detailwithin the image information. The focus merit value of the focussingscan line detector is normalised to the focus merit value of the imagingscan line detector, for example by dividing the focus merit valuesobtained at the detectors 2, 3 by the focus merit value obtained at theimaging line scan detector 2. By comparing the two values it is possibleto estimate in which direction the optimum focus level of the imagingdetector is to be found, and the focus of the apparatus is adjusted togive the imaging line scan detector a greater focus merit than thefocussing line scan detector.

In the case where the focussing line scan detector is positioned at afocus level 8 below the co-conjugate plane, when the focus merit of thefocussing scan line detector 3 gives a merit value less than the imagingline scan detector 2, then the optimum focus is either above the imagingline scan detector or the optimum focus is already at the imaging linescan detector (FIG. 3A, 3B). When the two focus merit values are similar(typically less than 5% difference) then the optimum focus is just belowthe imaging line scan detector (FIG. 3C). When the focussing line scandetector merit value is greater than the imaging line scan detectormerit value, the optimum focus is below the imaging detector (FIG. 3D).Using this information it is possible to keep the imaging line scandetector at or just above the optimum focus. If the focus distancebetween the two detectors is small enough then any error in focusposition produced by this method will be small enough that the qualityof focus will not be compromised.

In the above-described first embodiment of the invention, the focusmerit values between the imaging and focussing line scan detectors aresimply compared in order to estimate the in-focus level. The focus meritcurves seen in FIGS. 3A to 3D are for illustration purposes. Thecomparison protocol can be described as follows:

In general, if the focus merit of the imaging line scan detector isgreater than the focus merit of the focus detector by a predeterminedamount (typically 5%), move the focus level of the imaging line scandetector away from that of the focussing line scan detector (i.e. thein-focus level is closer to that of the imaging line scan detector thanthe focussing line scan detector). If the focus merit of the imagingline scan detector is smaller than the focus merit of the focussing linescan detector by a predetermined amount (typically 5%), move the focuslevel of the imaging line scan detector towards that of the focussingline scan detector. If the focus merit of the imaging line scan detectoris greater than the focus merit of the focussing line scan detector byless than the predetermined amount (typically less than 5% difference),move the focus level of the imaging line scan detector towards that ofthe focussing line scan detector.

It is to be understood that the “predetermined amount” may differdepending on the application. Preferably, the magnitude of thedifference in focus levels required in order to move the imaging linescan detector accordingly is nominally zero.

In a second embodiment of the present invention, the imaging line scandetector 2 and the focussing line scan detector 3 are both coloursensitive RGB detectors. This embodiment makes use of the residualchromatic aberration of the imaging lens 1. Each detector images thesame spatial region on the sample but at different focus levels, andeach red, green or blue channel within the detectors has a differentfocus level. Each channel on each detector calculates a focus meritvalue and the focus merits are then normalised to the channel on theimaging line scan detector 2 with the largest focus merit. This enablesmultiple points along a normalised focus merit curve to be plotted, asseen in FIGS. 4A to 4D.

The focus merit values obtained from the different channels can simplybe compared (without plotting a focus merit curve) in order to estimatethe in-focus level. For example, FIG. 4B shows the imaging line scandetector at the in-focus level for the green channel. This can bedistinguished from the imaging line scan detector being below thein-focus level (seen in FIG. 4A), because in FIG. 4B the merit valuesfor the blue channel on both detectors are similar, whereas this is notthe case in FIG. 4A. The protocol for comparing merit values for the RGBchannels can be described as follows:

(i) Measure the focus merit value of each channel on both detectors.

(ii) Select the two largest imaging line scan detector focus meritchannels.

(iii) Select the imaging line scan detector channel with the largestfocus merit as the primary channel and the other channel as thesecondary channel.

(iv) Normalise the focus merit of each channel to the primary imagingline scan detector channel focus merit value.

(v) If the primary focus merit of the imaging line scan detector isgreater than the primary focus merit of the focussing line scan detectorby a first predetermined amount (for example 5%), move the focus levelof the imaging line scan detector away from that of the focussing linescan detector.

(vi) If the primary focus merit of the imaging line scan detector issmaller than the primary focus merit of the focussing line scan detectorby the first predetermined amount, move the focus level of the imagingline scan detector towards that of the focussing line scan detector.

(v) If the primary focus merit of the imaging line scan detector isgreater than the primary focus merit of the focussing line scan detectorbut the difference is smaller than the first predetermined amount, andthe secondary focus level of the focussing line scan detector is betweenthe focus levels of the two detectors and the secondary focus merit ofthe imaging line scan detector is less than the secondary focus merit ofthe focussing line scan detector, then move the focus level of theimaging line scan detector towards that of the focussing line scandetector.

(vi) If the primary focus merit of the imaging line scan detector isgreater than the primary focus merit of the focussing line scan detectorbut the difference is smaller than the first predetermined amount andthe secondary focus level of the imaging line scan detector is betweenthe focus levels of the two detectors and the secondary focus merit ofthe imaging line scan detector is greater than the secondary focus meritof the focussing line scan detector, then move the focus level of theimaging line scan detector towards that of the focussing line scandetector.

FIG. 5 illustrates a schematic diagram of an apparatus 200 according toa third embodiment of the present invention. Similarly to the firstembodiment, an imaging line scan detector 2 is used to image lightoriginating from a sample (not shown) though lens 1. Typically thesample is moved relative to the system 200 in a plane perpendicular tothe optic axis of the imaging line scan detector 2 such that the sampleis imaged as a series of line scans. A first beam splitter 6 is used todirect light onto a first focussing line scan detector 3 as in the firstembodiment. However, the presently described third embodiment furtherincludes a second focussing line scan detector 9 which receives lightfrom a second beam splitter 11.

Beam splitter 6 produces a co-conjugate plane 7 a and beam splitter 11produces a co-conjugate plane 7 b. As in the first embodiment, the firstfocussing line scan detector is positioned below the co-conjugate plane7 a. The second focussing line scan detector 9 is positioned above theco-conjugate plane 7 b, as seen in FIG. 5 (i.e. the co-conjugate plane 7b is positioned between the second focussing line scan detector and thesample).

Due to the beam splitters, each imaging detector 2, 3, 9 simultaneouslyimages the same spatial location of the sample. Due to the presence ofthe second focussing line scan detector 9, three focus merit values canbe simultaneously calculated at three different focus levels—one foreach detector. The focus merit values are normalised to the focus meritvalue of the imaging scan line detector 2 (for example by dividing eachfocus merit value by the focus merit value obtained from the imagingline scan detector 2), and the three measurement values are used to plota graph of focus parameter (ordinate) in the form of normalised focusmerit values against focus level (abscissa). Such a “focus merit curve”is illustrated in FIGS. 6A-6C. The maximum of the focus merit curveprovides the in-focus level of the sample by intersection of the curvemaximum with the abscissa, and the imaging line scan detector is movedtowards that maximum.

In a fourth embodiment, as an alternative to providing first and secondfocussing line scan detectors, the apparatus 100 schematicallyillustrated in FIG. 2 may further comprise a detector drive (not shown)operable to move focussing detector 3 to and fro along its optic axis 3a. This allows a range of focus merit values to be obtained at differentfocus levels above and below the co-conjugate plane 7. The focus meritvalues can then be normalised to those of the imaging line scandetector, and a focus merit curve generated using the data from thefocussing detector 3. The maximum of the focus merit curve indicates thein-focus level of the sample and the imaging line scan detector 2 ismoved towards the in-focus level in the same way as described above.

In a fifth embodiment of the present invention, first 3 and second 9focussing line scan detectors are positioned adjacent the imaging linescan detector 2, as shown in FIGS. 7A and 7B. The focussing line scandetectors are typically located either side of the imaging line scandetector, although this is not essential. In the presently describedfifth embodiment each of the line scan detectors is located within theimage plane 15 as illustrated in FIG. 7A. The image lens 1 is typicallyrotationally symmetric, which produces the circular image plane 15. Thetwo focussing line scan detectors are located at different focus levelsto the imaging line scan detector, and at different focus levels to eachother (clearly shown in FIG. 7B).

As the focussing line scan detectors 3, 9 receive image informationalong different optic axes than the imaging line scan detector 2, thisadvantageously means that each detector is fully illuminated. However,it also means that the spatial region of the sample imaged by detectors2, 3, 9 is different. This means that focus merit values obtainedsimultaneously temporally will be affected not only by the focus andfocus level, but also by the spatial content for each of the regionsimaged. This can be overcome by temporally shifting image data collectedfrom each of the detectors 2, 3, 9 such that image data from the samespatial region on the sample can be compared between the detectors 2, 3and 9. As can be seen in FIG. 7A, as the target moves relative to theline scan detectors (shown by arrow 8), light will impinge first ondetector 3, followed by detector 2 and lastly on detector 9. Using atime delaying process based on the scan speed, the same spatial regionfrom the target can be compared at the different focus levels providedby detectors 2, 3 and 9.

Although FIGS. 7A and 7B show two focussing line scan detectors, theskilled person will appreciate that one, or three or more, focussingline scan detectors may be used.

Often, due to the physical size of the line scan detectors and theirpackaging with respect to the image plane 15, it is not possible tolocate the focussing line scan detectors adjacent the imaging line scandetector as seen in FIG. 7A. With high magnification imaging systemsalthough the field numerical aperture may be high, the image numericalaperture is low and the conjugate length is long. This allows mirrors 5and 14 to be positioned off-axis to reflect image information tofocussing line scan detectors 3 and 9, as seen in FIG. 8 schematicallyillustrating apparatus 300 according to a sixth embodiment of theinvention. Here, “off axis” means off axis from the optic axis 2 a ofthe imaging line scan detector 2.

The mirrors 5 and 14 are preferably turning mirrors which are placed inthe beam path and direct the beam to off-axis focussing line scandetectors 3 and 9 but advantageously permit all light to impinge on theimaging line scan detector 2. This set-up is equivalent to placing thefocussing scan line detectors adjacent the imaging scan line detector,as illustrated by references 4 and 13 in FIG. 8, which show the virtualpositions of detectors 3 and 9 respectively if no mirrors were present.

In a similar manner to third embodiment of the invention, the focussingline scan detectors 3, 9 are at different focus levels to that of theimaging line scan detector 2. Focus merit values normalised to those ofthe imaging line scan detector 2 can then be used to generate a focusmerit curve to estimate the in-focus level as described above.

FIG. 9 illustrates apparatus 400 according to a seventh embodiment ofthe invention where only one focussing line scan detector 3 is used,where the focussing line scan detector 3 is at a different focus levelto that of the imaging line scan detector 2. Here the in-focus level ofthe sample can be estimated by comparing normalised focus merit valuesof the imaging and focussing line scan detectors in the same manner asfor the first embodiment of the invention.

FIG. 10 schematically illustrates apparatus 500 according to an eighthembodiment of the present invention. In a similar manner to thatdescribed above, a turning mirror 5 is positioned off-axis from theoptic axis 2 a of the imaging line scan detector 2 and reflects imageinformation from the sample (not shown) to a focussing line scandetector 3. The apparatus further comprises a detector drive (not shown)operable to move the focussing line scan detector 3 to and fro along itsoptic axis 3 a. This movement is illustrated by the double headed arrow16. By moving the focussing line scan detector 3 along its optic axis 3a in this manner, a plurality of focus merit values can be obtained atdifferent focus levels. As explained above, these focus merit values aretemporally shifted such that focus merit values from the focussing linescan detector 3 can be compared with the focus merit value from theimaging line scan detector 2 with respect to the same spatial region onthe sample. The focus merit values from the focussing line scan detector3 are then normalised to the focus merit value from the imaging linescan detector 2 and a merit focus curve is produced using these data, asseen in FIG. 11. The nominal in-focus level of the sample can beestimated from the focus merit curve by the intersection of the curvemaximum with the abscissa, and the imaging line scan detector movedtowards that focus level. Typically at least three data points aredesirable to generate an acceptably accurate focus merit curve. Thenormalisation of the focus merit values from the focussing line scandetector 3 to that of the imaging line scan detector 2 is optional, andmay not be required if the sample is substantially homogenous.

One problem with the apparatus 500 of the eighth embodiment of thepresent invention is that the distance that the focussing line scandetector 3 has to move in order to alter the focus is scaled to thedepth of field by the square of the optical magnification. As anexample, in a system with an optical magnification of 40×, a 1 μm changein the field focus produces a 1.6 mm change in the focus position of thefocussing line scan detector 3. In a ninth embodiment 600 of the presentinvention, the turning mirror is replaced with a rotating turning mirror17 which rotates about turning point 20 where the principle ray of thefocussing line scan detector 3 intersects with the turning mirror 5.This apparatus 600 is schematically shown in FIG. 12.

The rotation of the turning mirror 17 causes the image to scribe an arc18 centred on the point 20. The focussing detector 3 remains stationary.This means that the focussing line scan detector has a different part(spatial location) of the sample imaged onto it as a result to thisrotation of the turning mirror 17, but because the image plane 19remains tangential to the scribed arc 18, the focus level of the samplebeing imaged by the focussing line scan detector 3 is altered. If therotation of the turning mirror 17 is synchronised with the motion of thesample 8 then the sample spatial location can be maintained on the focusdetector whilst the focus is altered during the turning process. Thiswill then enable a focus merit curve to be generated from the samespatial location, which advantageously removes sample effects from thefocus merit values. Once the curve has been generated the turning mirror17 can be set back to the original angle and the process repeated for anew measurement. As described above the focus merit curve can be used todetermine the in-focus level of the sample.

In a tenth embodiment 700 of the present invention (shown schematicallyin FIG. 13), the turning mirror 17 is rotated about a point 23 displacedfrom the intersection of the principle ray with the turning mirror 20.This means that a greater change in focus level of the image at thefocussing line scan detector 3 is produced for the same turning angle ofthe mirror, as seen in FIG. 13. This is because the rotation produces afocus change not only with the tangent 19 on the inscribed arc 18centred on the rotation point 23 but also with the displacement alongthe arc 18.

FIG. 14 shows an apparatus 800 according to an eleventh embodiment ofthe present invention. The arrangement is similar to that seen in FIG.10; however the focussing line scan detector is rotated about an axis 21perpendicular to the line (optic axis) of the focussing line scandetector 3. The focussing line scan detector 3 therefore produces adifferential focus 22 along the line of the detector 7 as schematicallyshown in FIG. 15. In an alternative embodiment, the focussing line scandetector is tilted with respect to its optic axis to effect thedifferential focus along the line of the detector. If the sample ishomogeneous in spatial frequency (detail) along the line of thefocussing line scan detector 3, then the focus merit function of thefocussing line scan detector 3 will peak along the length of thefocussing line scan detector where the in-focus plane intersects thefocussing line scan detector 3.

An example of this is in FIGS. 16A to 16C. In FIG. 16A the focussingline scan detector intersects the sample in-focus plane at approximately−5 on the scale along the focussing line scan detector 3. This is wherethe focus merit curve peaks, indicating that the imaging line scandetector 2 is below the in-focus plane (i.e. between the sample and thein-focus plane). FIG. 16C illustrates the case where the imaging linescan detector is above the in-focus plane (i.e. the in-focus plane isbetween the sample and the detector 2), and the focus merit curve peaksat approximately +5 on the scale along the detector 3. FIG. 16Billustrates the case where the imaging line scan detector is positionedat the in-focus plane.

If the sample is not spatially uniform, then this process (producing adifferential focus along the line of the focussing line scan detector 3)may give a misleading result. For example, if there is only detail onone side of the detectors 2 and 3 then even though the imaging detector2 may be at the correct focus level, the peak of the focussing line scandetector merit curve will be displaced from the centre of the focussingline scan detector 3 and biased towards the position of the detail.However, this situation can be rectified by using the image datacollected at the imaging line scan detector 2. In a twelfth embodiment,this image data can be used to calculate “detail merit” values in asimilar manner to the focus merit values obtained from the focussingline scan detector image data. The “detail merit” is the same focusparameter as the focus merit obtained from the focussing line scandetector. Therefore the detail merit values can be seen to be anumerical value dependent on the amount of fine detail in the imageinformation in the same way as the focus merit values. In alternativeembodiments the detail merit is a different focus parameter to the focusmerit and is normalised to the focus merit values obtained by thefocussing line scan detector.

These detail merit values can be weighted with the focus merit valueswhich will provide a corrected merit function giving the correct focusreading and preventing incorrect focus measurements.

FIGS. 17A to 17C show an example of the use of the detail merit values.Here, there is much more detail in the left hand side of the image, andthe detail merit curve therefore peaks on the left hand side of thedetector. The focus merit curves correspond to the focus merit curvesseen in FIGS. 16A to 16C, and relate to whether the imaging line scandetector is at, above or below the in-focus plane. However, due to thedetail being in the left side of the image, the measured focus meritpeak (generated using focus merit values from the focussing line scandetector 3) peaks on the left side of the focussing line scan detector 3even when the real focus peak is on the right side of the focussing linescan detector (see FIG. 17C). This means that if the measured focusmerit values were to be used directly, the system could even measure thewrong direction for optimum focus of the imaging line scan detector 2.

If the detail merit values are used—for example division of the measuredmerit by the detail merit—it is possible to recover the focus merit thatwill give the correct focus position. For example, as can be seen inFIG. 17B, the measured merit and the detail merit curves coincide at 0on the abscissa, which would give the correct focus position for theimaging line scan detector being in focus.

Features seen in any one of the above embodiments are not limited tothat single embodiment and may be used in any other embodiment.

1-42. (canceled)
 43. A method of estimating an in-focus level of atarget in an image scanning apparatus, wherein the image scanningapparatus comprises a first line scan detector configured to obtain oneor more image scan lines of the target and a second line scan detectorconfigured to obtain one or more focus scan lines of the target, themethod comprising: obtaining at least one image scan line of the targetusing the first line scan detector, each at least one image scan linebeing obtained at a respective focus level; obtaining at least one focusscan line of the target using the second line scan detector, each atleast one focus scan line being obtained at a respective focus level andwherein the focus level of the at least one image scan line is differentfrom the focus level of the at least one focus scan line; calculating atleast one focus parameter using at least the at least one focus scanline; and, estimating a nominal in-focus level of the target using thecalculated focus parameter(s).
 44. A method according to claim 43,further comprising the step of calculating at least one further focusparameter using either said at least one image scan line or a furthersaid focus scan line.
 45. A method according to claim 43, wherein thecalculating step comprises calculating at least one focus parameter foreach of the first line scan detector and second line scan detector usingthe respective at least one image scan line and at least one focus scanline.
 46. A method according to claim 43, wherein the step of obtainingat least one focus scan line comprises modulating a focus level of thesecond line scan detector such that a plurality of focus scan lines areobtained at different focus levels.
 47. A method according to claim 43,wherein the image and focus scan lines are obtained from a commonposition within a plane passing through the target and having a planenormal defining an optic axis along which each of the first and secondline scan detectors receive image information so as to produce the saidrespective image and focus scan lines.
 48. A method according to claim47, wherein image information is reflected to one of the said line scandetectors using a beam splitter.
 49. A method according to claim 43,wherein the image and focus scan lines are obtained from differentpositions in the target and wherein image information is obtained by thesaid first and second line scan detectors along different optic axesfrom the target so as to produce the said respective image and focusscan lines.
 50. A method according to claim 49, wherein imageinformation is reflected to one of the said line scan detectors using amirror.
 51. A method according to claim 50, wherein the minor is rotatedabout a point centred upon the respective optic axis so as to providefocus scan lines of the target at different focus levels.
 52. A methodaccording to claim 50, wherein the minor is rotated about a pointdisplaced from the respective optic axis so as to provide focus scanlines of the target at different focus levels.
 53. A method according toclaim 51, further comprising moving the target in accordance with therotation of the mirror such that the focus line scans are obtained froma common location upon the target.
 54. A method according to claim 43,further comprising moving the second line scan detector with respect tothe target so as to obtain a plurality of focus line scans at differentfocus levels.
 55. A method according to claim 43, further comprisingmodulating the focus level as a function of position across the scanline of the second line scan detector.
 56. A method according to claim43, further comprising using image data from one or each of the at leastone focus or image scan lines to generate a detail parameter; and usingthe detail parameter in calculating the focus parameter(s).
 57. A methodaccording to claim 43, wherein when the said line scan detectors aremulti-channel detectors, and the method comprises calculating anin-focus level for different channels of the detector.
 58. A methodaccording to claim 57, further comprising evaluating a focus parameterfor each channel and using one or more of the focus parameters for thechannels in the estimating step.
 59. A method according to claim 43,wherein the at least one focus parameter is a focus merit value having amaximum value representing an in-focus level.
 60. A method according toclaim 59, wherein the focus merit value is a normalised value.
 61. Amethod according to claim 59, wherein the focus level of the first linescan detector is modified by an amount and in a direction according themagnitude and sign of the difference between the focus merit value ofthe first and second scan line detectors.
 62. A method according toclaim 61, wherein if the said difference is greater than a firstpredetermined amount then the focus level of the first line scandetector is modified in a first sense.
 63. A method according to claim61, wherein if the said difference is less than a predetermined amountthen the focus level of the first line scan detector is modified in asecond sense.
 64. A method according to claim 43, wherein the methodfurther comprises adjusting the focus level of the first line scandetector to the nominal in-focus level.
 65. A method according to claim43, wherein a temporal shift is applied between the data from the scanlines of the first and second line scan detectors and wherein thetemporal shift is a function of the relative movement between the targetand the image scanning apparatus.
 66. A method according to claim 43,wherein image scan lines are obtained from a number of locations uponthe target so as to form a swathe.
 67. A method according to claim 66,wherein the focus level of the first line scan detector is adjusted tothe nominal in-focus level in real time during the formation of a swathesuch that the image scan lines within the swathe are obtained atdifferent focus levels.
 68. Image scanning apparatus comprising: a firstline scan detector configured to obtain one or more image scan lines ofa target; a second line scan detector configured to obtain one or morefocus scan lines of the target; and a processor configured to: obtain atleast one image scan line of the target at a respective focus levelusing the first line scan detector; obtain at least one focus scan lineof the target at a respective focus level using the second line scandetector and wherein the focus level of the at least one image scan lineis different from the focus level of the at least one focus scan line;calculate at least one focus parameter using at least the at least onefocus scan line; and estimate a nominal in-focus level of the targetusing the calculated focus parameter(s).
 69. Image scanning apparatusaccording to claim 68, further comprising a first focussing deviceconfigured to modify the focus level between the target and the firstline scan detector, and wherein the processor is further configured to:operate the first focussing device to move the focus level of the firstline scan detector to the estimated nominal in-focus level.
 70. Imagescanning apparatus according to claim 68, wherein the image scanningapparatus further comprises: a target stage for retaining the target;imaging optics for causing an image of the target to be provided to thefirst and second line scan detectors; and a drive system for causing thefirst line scan detector to obtain image information from differentlocations on the target.
 71. Image scanning apparatus according to claim70, wherein each of the first and second line scan detectors is arrangedto image a common location upon the target and wherein the imagingoptics includes a beam splitter to direct part of the image informationfrom the target to the first line scan detector and part to the secondline scan detector.
 72. Image scanning apparatus according to claim 70,wherein the first and second line scan detectors lie upon differentrespective optic axes of the imaging optics.
 73. Image scanningapparatus according to claim 72, wherein the imaging optics includes amirror arranged to direct part of the image information from the targetto one of the first or second line scan detectors.
 74. Image scanningapparatus according to claim 73, further comprising a mirror driveadapted to rotate the minor so as to direct different image informationto the said line scan detector.
 75. Image scanning apparatus accordingto claim 74, wherein the mirror drive is operated in accordance with thedrive system such that the focus line scans are obtained from a commonlocation upon the target.
 76. Image scanning apparatus according toclaim 68, further comprising a detector drive adapted to move the secondline scan detector along its respective optic axis.
 77. Image scanningapparatus according to claim 68, further comprising a detector driveadapted to rotate the second line scan detector so as to modulate thefocus level as a function of position across the scan line of the secondline scan detector.
 78. Image scanning apparatus according to claim 68,further comprising a third line scan detector for providing furtherfocus scan lines.
 79. Image scanning apparatus according to claim 68,wherein one or each of the first and second line scan detectors is amulti-channel detector.
 80. Image scanning apparatus according to claim68, wherein the first and second line scan detectors are substantiallyidentical.
 81. Image scanning apparatus according to claim 68, whereinthe focus levels of the focus line scan and image line scan detectorsare independently controllable.
 82. Image scanning apparatus accordingto claim 68, wherein the apparatus is a virtual microscope.
 83. Acomputer program product comprising program code means adapted in use toperform the method according to claim 43.